Monoselenophosphate (SeP) has been identified as the biological selenium donor for specific insertion of selenium into enzymes and tRNAs. In tRNAs a 2-thiouridine residue is converted to 2-selenouridine.. The enzyme that catalyzes this replacement reaction was isolated from Salmonella. The mechanism appears to involve a direct attack of SeP on the 2-carbon of 2-thiouridine with elimination of sulfur. No preliminary activation of the tRNA substrate is required. SeP synthesis; ATP + NaSeH SeP + Pi + AMP. Studies on the mechanism of this reaction were complicated by the presence of adenylate kinase, a trace contaminant in the enzyme preparations. Adenylate kinase was isolated from an overproducing E. coli strain and used to elicit antibodies in sheep. A polyclonal anti-adenylate kinase antibody affinity matrix was generated and used for further SeP synthetase purification. In the glycine reductase reaction Se-carboxymethylselenoprotein A is an intermediate in the conversion of the glycine to acetyl phosphate. An improved method for synthesis of [14C]CM-Se-selenoprotein A was developed using Tris [2-carboxyethyl] phosphine as reducing agent. This favored selective alkylation of the selenol group and higher reactivity as a substrate for protein C. [77Se]selenoprotein A was carboxymethylated with [13C]bromoacetate for 77Se NMR studies. Antibodies were prepared to C. sticklandii quinone-dependent p- nitrophenylphosphatase and used to detect the presence of this unusual enzyme in eukaryotes and other prokaryotes.